Cell culture
The WM115 and WM266-4 cells, as well as WM983A and WM983B cells, were established from a primary VGP melanoma and metastasis from the same patient, respectively [30]. In vitro, the cell lines with metastatic origin (WM266-4, WM983B) displayed a higher invasive potency, compared to cells from primary melanomas (WM115, WM983A), as assessed in 3D spheroids invasion assays [31] and human reconstructed skin models [32,33,34].
The WM266-4 and WM115 cells (obtained from the American Type Culture Collection) were grown in DMEM (Invitrogen, France) supplemented with 10% fetal bovine serum (Sigma, France), 2 mM glutamine, 100 UI/mL penicillin–streptomycin, and in a 5% CO2 atmosphere. The WM983A and WM983B cells (purchased from the Coriell Institute) were grown in MCDB153 medium with 20% Leibovitz’s L-15 medium (v/v), 2% FBS heat-inactivated (v/v), 5 μg/mL insulin and 1.68 mM CaCl2. The numerations of viable cells were performed using an Automated Cell Viability Analyzer (Beckman Coulter Vi-Cell).
Establishment of stable cell lines
WM266-4 cells were seeded at 6 × 105 cells in 60 mm dishes and transfected 24 h later using Lipofectamine 2000 (Invitrogen) with 1 µg of the pCMV6-PCDHB15 plasmid (DDK-tagged PCDHB15, RC207719, CliniSciences) or the pCMV6-MOCK plasmid corresponding to the same plasmid without the PCDHB15 cDNA sequence (obtained from the pCMV6-PCDHB15 plasmid by digestion with by EcoRI and XhoI, and self-ligation with a linker). The selection of transfected cells was performed in a medium containing 0.8 mg/mL of Geneticin (Gibco). Cell lines expressing PCDHB15 were established from 3 of 15 isolated clones. PCDHB15 expression was characterized by RT-qPCR. The control cell line (WM266-4 MOCK) is a pool of transfected cells with the pCMV6-MOCK plasmid. Transfected cells were maintained in culture in a medium containing 0.6 mg/mL Geneticin for 10 passages. These modifications did not impact morphology proliferation and viability. All experiments were conducted under 20 cell passages in culture.
Tumor samples
Tumor samples from four melanoma patients were retrieved from the tumor tissue bank at the Department of Pathology, IUCT-O Toulouse Hospital (France). The study was carried out in accordance with the institutional review board-approved protocols (CRB, AC-2013-1955), and the procedures followed the Helsinki Declaration. Pathological specimens consisted of frozen samples from primary (n = 13) and metastasis samples (n = 9). Additional frozen primary melanoma samples (n = 5) were provided by the Department of Experimental Oncology, European Institute of Oncology, Milan (Italy).
Cells treatment with 5-aza-2′-deoxycytidine (5azadC)
5-aza-2′-deoxycytidine (5azadC, decitabine) was bought from Sigma-Aldrich (France) and dissolved in acidic water at 10 mM and stored in single-use aliquots at − 20 °C.
WM266-4 cells were seeded at the density of 6 × 106 cells per 75 cm2 flasks (day 0) and treated with 5azadC after a 12 h period to allow cell attachment and synchronization in G0/G1 phase. Cells were treated daily for 72 h (day 1, 2, 3) at the indicated concentration of 5azadC. They were collected at day 4 for analysis of DNA methylation patterns by pyrosequencing and day 7 for expression analyses.
Genomic DNA isolation
Genomic DNA from cell lines was isolated using the DNeasy Tissue kit (Qiagen, France). Genomic DNA from patients’ samples was isolated using the QiaAmp kit (Qiagen, France).
Bisulfite pyrosequencing
Quantitative DNA methylation analysis was performed by pyrosequencing of bisulfite-treated DNA as previously described [35]. Sequences including CpGs were amplified using 20 ng of bisulfite-treated human genomic DNA and 5–7.5 pmol of forward and reverse primer, one being biotinylated. Two pairs of PCR primers were designed for PCR1 (CpG 1, 2, 3 and 4) and PCR2 (CpG 5 and 6). PCR was designed around the hypermethylated probes from previous Illumination 450 k Bead Chip analysis [21].
PCR1: Biotin-TTTAGAGTTGGTGTTGGATATAGAA (Forward) and CCAAAACCAAAATAAAAATCTAAAC (Reverse);
PCR2: TTTAGATTTTTATTTTGGTTTTGGA (Forward) and Biotin-TATAATATCTCTCCATTTATCCCAATATCT (Reverse).
Reaction conditions were 1 × HotStar® Taq buffer (Qiagen) supplemented with 1.6 mM MgCl2, 100 μM dNTPs and 2.0 U HotStar Taq polymerase (Qiagen) in a 25 μL volume. The PCR program consisted of a denaturing step of 15 min at 95 °C, followed by 50 cycles of 30 s at 95 °C, 30 s at 60 °C and 20 s at 72 °C, with a final extension of 5 min at 72 °C. A total of 10 μL of PCR product was rendered single-stranded as previously described and 4 pmol of the respective sequencing primers were used for analysis. Quantitative DNA methylation analysis was carried out on a PSQ 96MD system with the PyroGold SQA Reagent Kit (Qiagen) and results were analyzed using the PyroMark software (V.1.0, Qiagen).
TCGA DNA methylation data analysis
The TCGA-SKCM DNA methylation data was downloaded from GDAC Firehose Broad [36] on February 2021. Normalized beta values for the Illumina probes nearby the PCDHB15 gene were selected for comparative analyses. DNA methylation for primary melanoma (PRM), lymph node metastasis (LNM), and distant organ metastasis (DOM) was summarized using the mean value and the standard error of the mean. Differential DNA methylation was assessed by the Wilcoxon Rank-Sum test in R. All p values from multiple comparisons (> 50 tests) were corrected using the False discovery rate (FDR) method. The R/ggplot2 package was used for data visualization.
mRNA quantification
RNA was purified using the RNeasy Mini Kit (Qiagen, France) and quantified on a NanoDrop2000 (Thermo Fisher Scientific).
Quantification of PCDHB15 mRNA was performed by RT-qPCR. Total RNA (2 µg) was reverse transcribed into cDNA with the iScript cDNA Synthesis Kit (Bio-Rad, USA). Real-time PCR was performed according to the manufacturer’s recommendations, using SsoAdvanced™ SYBR® Green Supermix (Bio-Rad). The primers were: AGCAGGCCGAGCTCAGATTA (forward) and ATTGGCGTCCAAGACCAAGA (reverse). A CFX384 Touch™ Real-Time PCR Detection System from Bio-Rad (Marnes-la-Coquette, France) was used to run the following PCR program: 95 °C 10 min followed by 40 cycles of 15 s at 95 °C, 30 s at 65 °C for elongation, ended with a fusion cycle to determine the Tm of each amplification product.
The PCR data were analyzed with the CFX Manager v3.0 software (Bio-Rad) to generate the Ct values. The following quality controls were applied: amplification of a single product, no amplification in the NRT (No reverse transcription) condition, efficiency close to 100%, R2 > 0.98 and SD between technical triplicates < 0.3. The 2 − ΔΔCt method was used to generate the gene expression ratios by amplification of TBP (TATA box binding protein) TTGACCTAAAGACCATTGCACTTCGT (Forward) and TTACCGCAGCAAACCGCTTG (reverse) as normalizing control and data were presented as mRNA fold change of target RNA.
Western blot analysis
Total protein extract was obtained from confluent cells grown in 75 cm2 flasks. The cells were lysed in protein extraction buffer (10 mM Tris HCl, 120 mM NaCl, 1% NP40, 1 mM EDTA, 1 mM DTT and 1X proteases inhibitor (Complete™, EDTA-free Protease Inhibitor Cocktail, Sigma-Aldrich)). Samples were separated on 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes. After saturation with 5% dry milk in Tris NaCl 1% Tween 20, membranes were incubated with either anti-PCDHB15 antibody (NBP1-87322, Novus Biologicals), anti-DDK antibody (4C5, TA50011, OriGene) (1/1000 diluted in 5% dry milk in Tris NaCl 1% Tween 20) or anti-β actin antibody (MAB1501, Millipore, 1/1000 in 5% dry milk). After washes, the membranes were revealed with secondary HRP-coupled antibodies (Sigma-Aldrich). The signals were detected by ECL for β-actin (GE Healthcare) and Immobilon Western HRP Substrate (Millipore) for PCDHB15 and DDK. The chemoluminescent signals were acquired with a G:BOX imaging system (Syngene).
PCDHB15 cell surface expression
The expression of PCDHB15 at the cell surface was analyzed by flow cytometry. Cells were detached with 2 mM EDTA in PBS and incubated for 45 min at 4 °C with 1 µg/mL of anti-PCDHB15 antibody (NBP1-87322, Novus Biologicals) in PBS supplemented with 1% BSA. Cells were washed, counterstained with Alexa-647-conjugated goat anti-rabbit Ig antibodies (Invitrogen) and incubated with 0.5 mg/mL DAPI (Sigma). PCDHB15 expression was monitored on live cells (gated as DAPI-negative cells) on a LSRII flow cytometer using the Diva software (both from BD Biosciences, Le Pont-De-Claix, France).
3D cell invasion assay
WM266-4 cells were seeded in 96-well plates coated with agarose 1% (Sigma-Aldrich) in PBS (3000 cells in 100 µL medium per well). After 2 days at 37 °C in a 5% CO2 atmosphere, cells from one spheroid with a diameter of approximately 300 µm. For each condition, six spheroids were individually embedded in EMEM media (Lonza) containing 1% of bovin collagen I (BD Biosciences) and 2% SVF. Bright-field images from the initial spheroids were acquired with an Axiovert 200 M device (5X Plan-Neofluar objective, Carl Zeiss, Germany). After 24 h at 37 °C, spheroids were labeled 1 h with 2.5 µM calcein (calcein AM, BD Pharmingen) in PBS and fluorescent 6 z-stack images with 20 µm intervals were acquired. The fluorescent pictures were stacked and the total sizes of the spheroids were measured using the Image J (NIH) software. Invasion areas were obtained by subtracting the initial size of the spheroid. The invasion index represents the invasion area at 24 h normalized to the initial spheroid area. If cytotoxic effects appear, the initial spheroid area decrease and the data are not considered.
Aggregation assay
Cells were dissociated with 2 mM EDTA in PBS and seeded in a CELLSTAR® Cell-Repellent surface 96-well plate (Greiner Bio-One) (500 cells in 100µL medium per well), then centrifuged at 200 g for 8 min and left at rest for 45 min before time-lapse experiments. Time-lapse video microscopy images were acquired over 20 h (1 acquisition/15 min), by using an inverted widefield Zeiss Axio Observer Z1 microscope fitted with a 0.3 N.A 10 × objective and a CoolSNAP CDD camera (Roper scientific). At each time point and position, 5-µm spaced z-stacks in bright field were acquired using the Meta-Morph software. At each time point, and for each aggregate, areas of the cell aggregates were quantified using an algorithm developed on MATLAB software [37]. The aggregate areas were normalized to the calculated area at the beginning of time-lapse microscopy.
In vivo metastasis experiments
The animals were handled and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and European Directive EEC/86/609, under the supervision of the authorized investigators. Un-anesthetized 7-week-old female SCID mice (ENVIGO RMS SARL, Gannat, France) were injected into the tail vein with 3 × 106 viable cells in 200-μL PBS (WM266-4 WT, WM266-4-pCMV mock or each stable clone overexpressing the PCDHB15 gene). Groups were constituted of n = 15 animals for injection with mock, clone 8 and clone 12; n = 14 for clone 13. Twenty-one days after injection, mice were dissected and the organs (except brain) were visually inspected. Lungs only presented detectable metastases. They were recovered, formalin-fixed and paraffin-embedded. Sections were stained with hematoxylin and eosin (H&E). The number and area of metastasis were measured in whole lung sections by immunostaining with Tyrosinase antibody Mob299–05 (1/500) (Diagnostic BioSystems, Pleasanton, CA-USA). 3DHistech (Panoramic 250) was used to scan sections and measure metastases area. Statistics were performed using the Mann–Whitney test.